Towards an optimal sampling strategy for Alexandrium catenella (Dinophyceae) benthic resting cysts

The study proposes methodological developments to optimize sampling strategy of resting cysts of Alexandrium catenella to estimate their abundance with a predefined error. This work also aims to provide information on spatial distribution of resting cysts in sediments. The distribution mode of A. catenella resting cysts related to the abundance variability was studied through sediment cores sampling on four different spatial scales and using Ludox CLX gradient density method. The quantification method underestimates by a factor of 2 the resting cysts abundance in one gram of sediment. Application of Taylor's power law allowed us to define a compromise between sampling effort and abundance estimation error. In the case of A. catenella resting cysts from Thau lagoon, the optimal sampling strategy consists of sampling 10 stations on a surface of 2 km² for a given coefficient of variability (C) of 15%, sampling 3 sediment cores at each station (C = 30%) and counting only one replicate by core (C = 18%). Results related to the application of Taylor's power law are closely dependent on resting cyst density and aggregation in a given sediment. In our area, A. catenella resting cysts are mainly observed in the upper 3 cm of sediment. Horizontally, their heterogeneity is lower on 10 cm² surface and tends to stabilize itself beyond a surface of 10 m². Each author has to carry out this pre-sampling effort for his own resting cysts-forming species, in his own area, in order to increase accuracy of resting cyst mapping.     

Fungal surface protein mediated one-pot synthesis of stable and hemocompatible gold nanoparticles

Despite their large secretome and wide applications in bioprocesses, fungi remain underexplored in metal nanoparticles (MNP) biosynthesis. Previous studies have shown that cell surface proteins of Rhizopus oryzae play a crucial role in biomineralization of Au(III) to produce gold nanoparticles (AuNPs). Therefore, it is hypothesized that purified cell surface protein may produce in vitro AuNPs with narrow size distribution for biomedical and biocatalytic applications. However, different protein extraction methods might affect protein stability and the AuNP biosynthesis process. Herein, we have explored the extraction of cell surface proteins from R. oryzae using common detergents and reducing agent (sodium dodecyl sulfate (SDS) Triton X-100, and 1,4-dithiothreitol (DTT)) and their effect on the size and shape of the biosynthetic AuNPs. The surface proteins extracted with reducing agent (DTT) and non-ionic detergent (Triton X-100) produce spherical AuNPs with a mean particle size of 16 ± 7 nm, and 19 ± 4 nm, respectively, while the AuNPs produced by the surface protein extracted by ionic detergent (SDS) are flower-like AuNPs with broader size distribution of 43 ± 19 nm. This synthetic approach does not require use of any harsh chemicals, multistep preparation and separation process, favouring environmental sustainability. The biosynthetic AuNPs thus formed, are stable in different physiological buffers and hemocompatible, making them suitable for biomedical applications.     

The historical dendroarchaeology of two log structures at the Wynnewood State Historic Site,Castalian Springs,Tennessee, USA

Tree rings of eastern red cedar (Juniperus virginiana L.) were examined from cores extracted from two log cabins located at the Wynnewood State Historic Site in Castalian Springs, Sumner County, Tennessee. One cabin was reportedly built by the first explorer in the area, Isaac Bledsoe, sometime between 1772 and his death in 1793. The second cabin was known as Spencer's Cabin after the first settler of the region, Thomas Sharp Spencer, who lived in the immediate vicinity from 1776 to 1779. The goal of this research was to determine the probable construction year(s) for both cabins and determine whether Bledsoe and Spencer did indeed build these structures. Forty-one cores were extracted from Bledsoe's Cabin, and 30 were used for crossdating and building a floating chronology using COFECHA. The Bledsoe's Cabin chronology was then statistically and graphically crossdated using the eastern red cedar reference tree-ring chronology (ITRDB #TN031) from Norris Dam, Tennessee. We found a statistically significant correlation (r = 0.42, t = 4.18, n = 85, p < 0.0001) between the Bledsoe's Cabin chronology and the reference chronology, anchoring the chronology between 1720 and 1804, with nearly all cores indicating tree harvesting between February and April 1805. Twenty-two cores were extracted from Spencer's Cabin, and 17 were used to build a floating chronology for the cabin. Again, we found a statistically significant correlation (r = 0.44, t = 4.85, n = 100, p < 0.00001) with the reference chronology, which anchored the Spencer's Cabin chronology between 1726 and 1825. All trees appear to have been harvested between February and August in 1826. Therefore, neither structure was built by its historical namesake. No known historical documents suggest who the potential builders were, although the property was owned between ca. 1797 and 1829 by General James Winchester. He and his family, however, never resided on the Wynnewood property because Winchester had built a large multi-room structure in nearby Gallatin, Tennessee, by 1802.     

Enzyme-assisted extraction of lycopene from tomato processing waste

A central composite design was used to optimize the enzyme-assisted extraction of lycopene from the peel fraction of tomato processing waste. Tomato skins were pretreated by a food-grade enzyme preparation with pectinolytic and cellulolytic activities and then subjected to hexane extraction. The factors investigated included extraction temperature (10–50 °C), pretreatment time (0.5–6.5 h), extraction time (0.5–4.5 h), enzyme solution-to-solid ratio (10–50 dm³/kg) and enzyme load (0–0.2 kg/kg). Overall, an 8- to 18-fold increase in lycopene recovery was observed compared to the untreated plant material. From a response surface analysis of the data, a second-degree polynomial equation was developed which provided the following optimal extraction conditions: T = 30 °C, extraction time = 3.18 h and enzyme load = 0.16 kg/kg. The obtained results strongly support the idea of using cell-wall degrading enzymes as an effective means for recovering lycopene from tomato waste.     

Preparing micro sections of entire (dry) conifer increment cores for wood anatomical time-series analyses

The type of samples most commonly used in dendro sciences are increment cores of conifers. These cores allow for an easy determination and measurement of ring-width variations over long time periods. For wood anatomical analyses, the cores have to be split into pieces to enable the preparation of high quality micro sections for detailed measurements of cell properties. A major drawback of this procedure is the fact that it is labor intensive and time consuming. We present a new technique enabling the preparation of micro sections of entire increment cores up to a length of 40 cm. For that purpose we combined standard wood-anatomical techniques with the application of Mowiol glue and common Tesa tape. We tested the introduced method on increment cores of Larix decidua Mill. sampled years ago for ring-width analyses to reanalyze them on a microscopic level. The ability to cut these long sections will tremendously reduce the time needed to prepare micro sections. This is of special interest for wood anatomical image analyses of cores used before to create long ring-width chronologies for any kind of environmental reconstructions.     

The DNA–DNA spacing in gemini surfactants–DOPE–DNA complexes

Gemini surfactants from the homologous series of alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, number of spacer carbons n = 2 – 12) and dioleoylphosphatidylethanolamine (DOPE) were used for cationic liposome (CL) preparation. CLs condense highly polymerized DNA creating complexes. Small-angle X-ray diffraction identified them as condensed lamellar phase L_α^C in the studied range of molar ratios CnGS12/DOPE in the temperature range 20 – 60 °C. The DNA–DNA distance (d_DNA) is studied in dependence to CnGS12 spacer length and membrane surface charge density. The high membrane surface charge densities (CnGS12/DOPE = 0.35 and 0.4 mol/mol) lead to the linear dependence of d_DNA vs. n correlating with the interfacial area of the CnGS12 molecule.     

High yield preparation of ganglioside GM1 using recombinant sialidase from Cellulosimicrobium cellulans

Cellulosimicrobium cellulans employs extracellular sialidase to selectively convert polysialogangliosides to ganglioside GM1. We cloned this novel sialidase gene (ccsia) from C. cellulans sp. 21, and overexpressed recombinant sialidase (CcSia) protein in E. coli BL21 (DE3) by high cell density fermentation. The presence of an N-terminal hexa-His tag allowed for purification using nickel affinity chromatography (2.3-fold, specific activity 41.5 U/mg). As determined by gel electrophoresis and gel filtration chromatography, the molecular weight of CcSia was found to be about 75 kDa, consistent with sequence analysis (75,271 Da). CcSia transformed polysialogangliosides GD1a, GD1b and GT1b into GM1. For this reaction, the response surface approach showed that optimal conditions in a 1-L system were 2 h incubation at 32.5 °C and pH 5.2, with substrate concentrations of 10 g/L and crude enzyme concentration 1 g/L, respectively. Under above conditions, 10 g/L of ganglioside was completely converted to the product GM1 with a yield of 52%. Our studies demonstrate CcSia could be used for industrial preparation of ganglioside GM1 by the pharmaceutical industry.     

Frettes (Haute-Saône,France) : un gisement de plein-air du paléolithique moyen,premiers résultats

Frettes is an open air site discovered in 1900 by Docteur Bouchet. Since that date, the site has been prospected by several other local researchers. The surface industry condensed by areas of 50 m², shows something defined around a Ferrassie Mousterian with oriental similitudes. Following a sondage campain in 1988 and 1989, the site has been dug in 1990 and 1991 by Gilles Huguenin. This excavation spread over 48 m² and 3746 artefacts constitute one main lithic serie with about 78 artefacts per meter. During the first polls of 1988-1989 of 4 m² of extension and the fulfilment of a 200 m trench which had for aim to compare polls all together, the lithic obtained was rich of Levallois finds (cores, flakes, tool-flakes), blade finds (rare cores, blades, blade-tools). During the excavation of 1991-1991, the lithic inventory shows the same typology and technology characteristics that we can describe as following: a main Levallois composition and in a lesser extent a blade composition. For the blade part of the lithic serie, we can notice the important part of blade cores done on the tranche of nodules or flakes and the great part of ventral flake realization next to the tradition flat or volume cores. During those researches, the question of a single or recurrent occupation was naturally written. The spatial analysis pleads for one main occupation. The blade artefacts commonly show the same characteristics than the Levallois artefacts position and density: common areas of high density, common areas of lower density, decline areas from higher density to the lowest. The archaological level shows a short vertical dispersion, around 7 cm, the maximum is 12 cm located on plots where the chalk records accidents. Retouched tool are made by noches, simple scrapers, double scrapers, few burins and end-scrapers. Tools are also composed of truncatures and blade convex scrapers. The typology of the flakes is in accordance with types of cores: Levallois flakes, blades or allongated flakes, crested-blade. Located on the east side of a great chalky depression, the stratigraphy of the site can be very poor, away or can be developed with more than 2 meters of deposits before to find the rock. The raw material is local, the blue and white flints are coming from the Bajocien system. Other minerals are also present, they are non-silex like quartz or quartzite coming from the Saône river and his area far from 20 km from the site. This stratigraphy also permits us to propose a first model of major phase of deposit and major phase of frequentation of the landscape by hominids. This article underlines the first totally new results of the stratigraphy, the interpretative results and show us the main characters of an industry composed by Levallois and blade methods and where refittings are numerous and do reflect all debitage steps.     

Nitrogen rhizodeposition assessed by a NH3 shoot pulse-labelling of Lolium perenne L. grown on soil exposed to 9 years of CO2 enrichment

The effects of elevated CO₂ concentration upon rhizodeposition of nitrogen were investigated on field-grown Lolium perenne planted in soil cores set into the resident soil of a intensively managed ryegrass sward treated with elevated CO₂ for nine consecutive years, under two contrasted N fertilisation regimes (Swiss FACE Experiment). The planted cores were excavated from the ambiant (35 Pa pCO₂) and enriched (60 Pa pCO₂) rings at two dates during the growing season (spring and early autumn). The cores were brought back to the laboratory for a pulse-labelling of ryegrass shoots with ¹⁵NH₃, in order to quantify ¹⁵N-rhizodeposition.A recovery of 10–16% of the total ¹⁵N administred to the plant was recovered in the plant–soil system 48 h after the pulse-labelling; significant amounts of ¹⁵N were released into the soil adhering (adhering soil: AS) to the roots (0.44 μg ¹⁵N g AS⁻¹ and 0.60 μg g AS⁻¹ in the spring and the autumn samplings, respectively).In the spring sampling, there was no effect of atmospheric CO₂ concentration on N rhizodeposition. In the autumn sampling, elevated CO₂ stimulated N rhizodeposition that amounted to 7.2 and 5.2 mg ¹⁵N m⁻², under elevated and ambient CO₂, respectively. Nitrogen rhizodeposition was higher at high N (56 gN m⁻²) than at low N fertilisation (14 gN m⁻²), whatever the sampling date investigated.The mechanisms by which elevated atmospheric CO₂ leads to a stimulation of the net root-released N flux remains to be investigated: was it caused by a higher nitrogen immobilisation by the microbial biomass and a reduced re-assimilation of mineralized N and/or by a stimulation of N efflux from roots? Concomitant to the observed reduction of C rhizodeposition, the stimulation of net N efflux suggests that the quality of root released compounds was modified under elevated CO₂ concentration.     

Environmental and climatic changes in the central Mediterranean Sea (Siculo–Tunisian Strait) during the last 30 ka based on dinoflagellate cyst and planktonic foraminifera assemblages

A high resolution micropalaeontological study of the core MD 04-2797 CQ recovered in the Sicilian–Tunisian Strait provides insights into the paleoclimatic history of the Mediterranean Sea at the transition between the western and eastern basin over the last 30 ka. Using the analysis of dinoflagellate cyst and planktonic foraminiferal assemblages, we reconstruct the paleoenvironmental changes that took place in this region. High abundances of cold temperate dinocyst species (Nematosphaeropsis labyrinthus, Spiniferites elongatus, Bitectatodinium tepikiense) and the polar planktonic foraminifera Neogloboquadrina pachyderma (left coiling) reveal three major cooling events synchronous with North Atlantic Henrich events 1 and 2 (H1 and H2) and the European and North Atlantic Younger Dryas event. During the Holocene, the presence of warm dinocyst species (Spiniferites mirabilis and Impagidinium aculeatum) and planktonic foraminifera (Globorotalia inflata and Globigerinoides ruber), reflects a significant increase of sea surface temperatures in the western Mediterranean basin, but a full warming was not recorded until 1500 years after the onset of the Holocene. Moreover, our results show that the Holocene was interrupted by at least four brief cooling events at ~ 9.2 ka, ~ 8 ka, ~ 7 ka and ~ 2.2 ka cal. BP, which may be correlated to climatic events recorded in Greenland ice cores and in the Atlantic Ocean.     

Development of an enzymatic synthesis approach to produce phloridzin using Malus x domestica glycosyltransferase in engineered Pichia pastoris GS115

Phloridzin has been an attractive target for the development of preparation methodologies due to its various applications as pharmaceutical ingredient and food additive. In this study, an enzymatic synthesis approach using engineered P. pastoris GS115 was developed to produce phloridzin. Four factors, identifying as key factors affecting the phloridzin yield, were optimized by response surface methodology as follows: induction time 121.8 h, pH 7.0, methanol 0.75%, and a rotation speed 194 rpm. The maximal yield of phloridzin reached 41.59 mg/L (95.31 μM) and the specific bioconversion rate of substrate was up to 98.50% with 1L working volume in a 5L bioreactor under the optimal conditions. Phloridzin with purity of 93.98% and recovery of 78.14% was achieved just using affinity chromatography and preparative high performance liquid chromatography (Pre-HPLC). These results confirm the enzymatic synthesis developed provides an efficient alternative to traditional approach for the preparation of phloridzin in vitro.     

Simulation study to determine necessary sample sizes for image analysis-based quantitative wood anatomy of vessels of beech (Fagus sylvatica)

Quantitative analysis of wood anatomy is a powerful dendroecological tool to provide new insights into environmental signals encoded in tree-rings. Nevertheless, studies with long time series or large sample sizes are not very common for diffuse-porous tree species due to the laborious sample preparation and image analysis. Finding a compromise between sample size and accuracy of sample estimators is therefore of crucial importance. In this simulation study, we present a subsampling method, which can make the quantitative analysis of wood anatomy of European beech more efficient. Based on our study material we demonstrate that for the parameters vessel size, vessel density, total vessel area and accumulated hydraulic conductivity the environmental signals are captured with high trueness (deviation < 5%) when analysing only a 1 mm wide radial stripe along the increment core. Nevertheless, when information about vessel grouping indices is required, for high trueness of the results a wider radial stripe of ∼2.2 mm needs to be analysed.     

Innovative metal oxide-based substrates for DNA microarrays

In the present report, we propose a novel approach to synthesize DNA microarrays that employs immobilization of the nucleic acid molecules onto zinc and iron oxide surfaces through their phosphate backbone. Oxide films were prepared by the sol–gel technique and the resulting surfaces were characterized especially with respect to surface chemistry and morphological features by both X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). ZnO films annealed at T ? 300 °C show the most promising surface features to be employed for DNA microarray preparation, i.e. high density of binding sites (hydroxyl groups), smooth and homogeneous surfaces, high optical transmittance in the visible spectral range suitable for detection using fluorescence, and easy handling during preparation procedures. The analysis of nucleic acid retention on the oxide layers was performed by the scanning of dye-labelled DNA previously printed on the substrate using the DNA microarray robotic arm. Clearly visible spots with regular shape were revealed above the background noise indicating that anchoring of the DNA on the treated surface is efficient and does not interfere with hybridization processes. The use of suitably engineered zinc oxide film makes the immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.     

Immobilization of trypsin on polysaccharide film from Anacardium occidentale L. and its application as cutaneous dressing

Polysaccharide obtained from Anacardium occidentale L. gum was used for trypsin entrapment using cellulose (gaze) as a support and this preparation was applied as cutaneous wound healing. Trypsin release in vitro and the influence of pH and temperature on activity, stability and storage time of entrapped enzyme were evaluated. The preparation showed that it was still capable to release enzyme even after 48 h. Entrapped enzyme presented an optimal pH and temperature of 8.6 and 55 °C, respectively. Also, it was stable at high temperature (45 °C for 60 min) and wide range of pH, retaining 80% of its initial activity when stored for 28 days at 25 °C. Histopathological analysis of mice skin wound healing under the entrapped trypsin preparation treatment showed an acceleration of fibroblast proliferation, neovascularization of granulation tissue and stimulating effect on the epithelium formation compared to the skin wound under the treatment using preparations without trypsin. These results demonstrate that the trypsin–polysaccharide–cellulose preparation could be used in cutaneous dressing applications for wound healing.     

Late Quaternary (Weichselian) alluvial history and neotectonic control on fluvial landscape development in the southern Körös plain,Hungary

Four drill cores and a clay pit section have been examined in the southern part of the Körös plain to understand the history and controls on alluvial sedimentation for the last ~ 40 ka. Four facies groups were identified, such as channel, channel margin, floodplain and floodbasin with seven distinctive facies. Magnetic susceptibility and mineralogy have further characterized the sedimentary facies indicating shifts in humidity conditions, variations in sediment flux and pedogenesis. Detailed pollen analysis of a 7.5 m thick clayey succession indicated climatic variability within the MIS 3 period. The spatial distribution of the different facies allowed outlining alluvial architecture of the study area. Three depositional units composed of various facies were identified based on OSL and radiocarbon data. These packages correspond to three major phases of channel activity: (F-I) pre-LGM period (> 30 ka to 24 ka), (F-II) post-LGM interstadial (18–16 ka), and (F-III) Late Glacial < 15 ka to ~ 10 ka). The pre-LGM and post-LGM “interstadial” phases are characterized by meandering river patterns, while the Late Glacial fluvial activity is characterized by a braided system in the area. Higher sediment supply feeding this braided river was probably caused by neotectonic uplift of the southern margin of the basin, documented by a significant stratigraphic gap between 25 and 14 ka.     

Immobilization of lipase on epoxy-activated Purolite® A109 and its post-immobilization stabilization

In this study, Purolite^® A109, polystyrenic macroporous resin, was used as immobilization support due to its good mechanical properties and high particle diameter (400 μm), which enables efficient application in enzyme reactors due to lower pressure drops. The surface of support had been modified with epichlorhydrine and was tested in lipase immobilization. Optimized procedure for support modification proved to be more efficient than conventional procedure for hydroxy groups (at 22 °C for 18 h), since duration of procedure was shortened to 40 min by performing modification at 52 °C resulting with almost doubled concentration of epoxy groups (563 μmol g⁻¹). Lipase immobilized on epoxy-modified support showed significantly improved thermal stability comparing to both, free form and commercial immobilized preparation (Novozym^® 435). The highest activity (47.5 IU g⁻¹) and thermal stability (2.5 times higher half-life than at low ionic strength) were obtained with lipase immobilized in high ionic strength. Thermal stability of immobilized lipase was further improved by blocking unreacted epoxy groups on supports surface with amino acids. The most efficient was treatment with phenylalanine, since in such a way blocked immobilized enzyme retained 65% of initial activity after 8 h incubation at 65 °C, while non-blocked derivative retained 12%.     

Evaluating the potential for dendrochronological analysis of live oak (Quercus virginiana Mill.) from the urban and rural environment—An explorative study

Live oak (Quercus virginiana Mill.) is a long lived, broadleaf, evergreen species native to the lower Coastal Plain of the southern and southeastern United States and is frequently planted in the urban environment. However, live oak has received limited attention for its potential for dendrochronological analyses possibly because its growth-ring boundaries may be less defined due to the absence of a distinct dormancy period. The objective of this explorative study was to determine whether live oak rings in both urban and historic trees could be accurately identified and measured for dendrochronological applications. One hundred urban live oaks were assessed as well as four cross-sectional specimens from historic sites in the southeastern United States. Age of all cored live oak ranged from 7 to 29 (average = 16.8 rings), while cross-sections were taken from trees with an average age of 175 years. A chronology was created from cores that had the highest interseries correlations (n = 27), resulting in an average interseries correlation of 0.654. The analysis of live oak cross sections revealed that the rings on live oak are very faint and barely discernible, delimited mainly by a faint row of earlywood vessels with no or barely visible terminal parenchyma. Rings on these sections were erratic (i.e., fading out along the circumference) and non-concentric (i.e., a particular ring could be both wide and narrow at certain points along the circumference) so that any attempt to measure the rings on opposing radii would have produced meaningless results. However, based on the results of this study, we conclude that the dendrochronological analysis of live oak is possible with careful sample preparation and analysis and that results would be reliable, even for urban trees. Results of this study suggest that urban tree-ring series with higher interseries correlations were more vigorous than those with lower values, suggesting that urban environmental stresses related to individual sites may decrease the usefulness of crossdating techniques for evaluating annual rings on younger, urban trees.     

Purification and characterization of α-l-rhamnosidase from Penicillium corylopholum MTCC-2011

An extracellular α-l-rhamnosidase has been purified to electrophoretic homogeneity from the culture filtrate of Penicillium corylopholum MTCC-2011 using a simple procedure consisting of concentration by ultrafiltration and cation exchange column chromatography on carboxymethyl cellulose. The sodium dodesyl sulphate polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass of 67.0 kDa. The native – polyacrylamide gel electrophoresis analysis also gave a single protein band confirming the purity of the enzyme and also showing that the enzyme is a monomer in the native state. The K_m and k_cat values of the enzyme were 0.42 mM and 35.7 s^?1, respectively, using p-nitrophenyl α-l-rhamnopyranoside as the substrate. The pH and temperature optima of the enzyme were 6.5 and 57.0 °C, respectively. The purified enzyme preparation successfully hydrolyzed naringin and rutin to prunin and quercetin glucoside, respectively. Thus it can be used for the preparation of these pharmaceutically important compounds.     

Synthesis and characterization of a pH-sensitive conjugate of isoniazid with Fe3O4@SiO2 magnetic nanoparticles

The Letter describes the preparation and characterization of a conjugate of isoniazid (INH) with magnetic nanoparticles Fe₃O₄@SiO₂ 115 ± 60 nm in size. The INH molecules were attached to the surface of nanoparticles by a covalent pH-sensitive amidine bond. The conjugate was characterized by X-ray diffraction, SEM, dynamic light scattering, IR spectroscopy and microanalysis. The conjugate released isoniazid under in vitro conditions (pH = 4; 37 °C; t_1/2 ≈ 115 s). In addition, the cytotoxicity of the Fe₃O₄@SiO₂–INH conjugate was evaluated in SK-BR-3 cells using the xCELLigence system.